The methods disclosed herein relate to detecting and quantifying nucleic acids, especially small RNAs, including miRNAs and genes coding for small RNAs including but not limited to miRNAs. In the disclosure various reactions may be described in respect to miRNA, but it is understood by those skilled in the art that such reactions or reaction conditions would apply similarly to other small RNAs, of which miRNAs are an important subclass. More specifically, the present methods relate to novel reaction conditions under which a homopolymeric tail is added to the 3′ end of a small RNA using poly-adenosine (poly(A)) polymerase (polyadenylation) and subsequently rendered into cDNA by reverse transcriptase under conditions that permit greater discrimination of a target miRNA from non-miRNA contained in the same sample. These novel reaction conditions lead to greater specificity of addition of nucleotides by both polymerases and greater specificity of detection of the small RNA being assayed. These reaction conditions allow for convenient use of both poly(A) polymerase and reverse transcriptase under substantially the same ionic and buffer conditions, thereby avoiding undesired dilution of the sample or purification of products (with potential loss of desired materials) between the poly(A) polymerase reaction and the synthesis of a cDNA copy catalyzed by reverse transcriptase. The ability to forego all purification or substantial dilution of sample between these two reactions as well as subsequent reactions that measure miRNA amounts greatly increases the ease of the assay, as well as its reproducibility, accuracy and sensitivity.
MicroRNAs (miRNA) are single-stranded RNA molecules of about 18 to 28, often about 22, nucleotides in length, which regulate gene expression. Other small RNAs, including piRNAs, snoRNAs, and small guide or sgRNAs, play essential biological roles, and their detection and quantitation are desired. Still other small RNAs may be discovered that play important biological roles as well. Herein a small RNA is defined as an RNA shorter than about 100 nucleotides in length. All miRNAs, piRNAs, sgRNAs, snoRNAs and snRNAs, as well as other RNAs having fewer than 100 nucleotides are “small RNAs”. These RNAs are encoded by genes that are transcribed from DNA, but the transcription products are not translated into protein (non-coding RNA). They are, in the case of miRNAs, processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules generally are partially complementary to one or more messenger RNA (mRNA) molecules, and their principal regulatory function is to down-regulate gene expression by translation repression. They were first described in 1993 by Lee and colleagues and are today recognized as important regulatory molecules in eukaryotic cells. MicroRNAs have been shown to play key roles in development, apoptosis, and cancer. They have also been shown to be coded for by certain viruses.
Specific, sensitive and quantitative detection and assay of individual small RNAs and detection of miRNAs play an especially important role in biomedical and biological research and promise to be of diagnostic and prognostic value in medical practice as well.
Notwithstanding their importance as regulatory molecules, small RNAs represent a small fraction of the RNA species in a eukaryotic cell. Because they are short, in the case of miRNAs, about 18-28 nucleotides in length, or 18-50, or 18-100, and share homology or partial homology, with the genes they regulate, detection of specific miRNAs and especially accurate quantitation of miRNAs present experimental challenges. Improvements to methods for the specific detection and quantitation of miRNAs will accelerate basic research. Improved assays of miRNAs can prove of diagnostic or prognostic value in medicine.
One of the most widely used and effective ways to detect and quantify RNAs is to produce a complementary DNA (cDNA) transcript of specific RNA, and then to amplify that DNA using the polymerase chain reaction (PCR) under conditions where the accumulation of the amplified product is monitored during the amplification, usually by means of fluorescent molecules added to the PCR. This method is widely known as Real-Time Quantitative RT-PCR(RT-qPCR). Improved methods to assay specific miRNAs by RT-qPCR are desired.
When assaying for species as low in abundance as miRNA, the specificity of detection and accurate quantitation are usually higher priorities than absolute analytical sensitivity. Accordingly, modifications to standard processes that increase specificity and accuracy of quantitation, even if they result in minimal or modest losses of absolute analytical sensitivity, are likely to be preferred.
In light of the vast abundance of RNAs other than the specific small RNA being assayed, or especially in the case of assaying for a specific miRNA, non-specific amplification of other sequences can compromise the sensitivity, specificity, and precision of an RT-qPCR assay for a small RNA, especially an miRNA.
One approach to the specific amplification of miRNAs, but also applicable to other small RNAs, is that used by Ro et. al. (Biochem Biophys Res Commun (2006) 351 (3) 756-763) which involves addition to the 3′ prime end of the miRNA to be amplified (the target miRNA) a poly(A) tail of greater than about 20 base pairs. This can be done using poly(A) polymerase and ATP; this lengthens the miRNA molecule in order to facilitate subsequent cDNA synthesis and detection. An oligonucleotide, with the 5′ sequence region matching a universal qPCR primer sequence and the 3′ sequence region being approximately 20 d(T) bases and complementary to the poly(A) tail, is hybridized to the 3′ end of the target miRNA to provide a template which can be used by a reverse transcriptase to produce a cDNA molecule capable of being amplified by PCR. This cDNA contains a region complementary to a specific miRNA, a central region of approximately 20 T's (on one strand and the same number of A's on the other) and a third region that contains an arbitrary sequence that can serve well as a site for specific priming by a PCR primer, i.e., a universal primer. Note, that cDNAs made from different miRNAs, will have all have a common universal primer sequence. However, this reaction is not absolutely specific and other RNAs present in the sample and such other RNAs having been present in vast abundance relative to the targeted miRNA in the cells, from which the miRNA is prepared, in spite of earlier purification steps taken to eliminate them, may have poly(A) tails added to them. Some of these non-miRNAs may also serve as template for reverse transcriptase and receive a universal primer sequence. Reduction in non-specific tailing of RNAs and generation of RNAs other than miRNAs that contain universal primer sequence is desired. All of the advantages described above pertaining to miRNAs also apply to other small RNAs.
Purified poly-adenosine polymerase has been commercially available for years to artificially produce poly(A) tails on RNA molecules in vitro. Recently this enzymatic activity has been utilized as the first step in an miRNA cDNA generation system to increase the length of the miRNA for increased PCR performance and to provide a uniform primer binding site to initiate cDNA synthesis.
Purified reverse transcriptases have been used to produce DNA copies of RNAs in vitro following well accepted reaction conditions and protocols. This reaction has become a standard process used in thousands of research laboratories. Several manufacturers of research products have marketed kits containing premixed buffers for customer use. Like most enzymes that catalyze the polymerization of nucleotide triphosphates into DNA or RNA using a template nucleic acid as a template, reverse transcriptases have standard reaction conditions in which the concentration of magnesium ions is maintained between about 2 millimolar and 5 millimolar. Biochemists experienced in the optimization of reverse transcriptase reaction in vitro are especially careful to optimize the concentration of magnesium ions within this overall range, about 2 millimolar to about 5 millimolar. The Applicants are unaware of any published reports of reaction conditions for reverse transcription in vitro carried out with a magnesium concentration above 10 mM.
The concentration of cations, particularly divalent cations such as magnesium and manganese are known to affect the secondary structure of nucleic acids, particularly single stranded nucleic acids such as most RNAs found in cells, including human cells (Bukhman and Draper, J. Mol. Biol. (1997) 273, 1020-1031) Nearly all RNAs that are abundant in cells, especially, but not only ribosomal RNAs, have considerable secondary structure that can be affected by the ionic environment, especially the concentration of cations such as magnesium and manganese. While methods are known that result in the preferential purification of small RNAs, miRNA purified using these methods can be heavily contaminated with larger RNAs, with other small RNAs, and degradation fragments of large RNAs such as ribosomal RNAs. In any sample of RNA, even one enriched for shorter RNAs, any particular miRNA will be a minor constituent in the sample. Thus, there is a need in the art for improved methods which allow selective amplification of one or more target small RNA molecules in a sample.